The Sir2 (or sirtuin) protein family plays critical roles in aging, DMA repair, apoptosis, resistance to cell stress, and metabolism. However, the molecular basis for the varied phenotypes has remained elusive. The majority of sirtuins catalyze a reaction in which the cleavage of NAD+ and histone/protein deacetylation are coupled to the formation of O-acetyl-ADP-ribose (OAADPr), a novel metabolite. Several reports suggest that some sirtuins are mono-ADP-ribosyltransferases, while others have suggested that these enzymes harbor both activities. Detailed mechanistic studies have yet to validate these reports. The dependence on NAD+ and the generation of a potential second messenger offer clues toward understanding their cellular functions. Here, we will focus on answering questions of molecular mechanism, filling major gaps in our understanding of these unique proteins. To address their molecular and biological functions, this proposal outlines a multi-disciplinary approach, involving chemistry, enzymology and genetics. In Aim 1, the catalytic pathways that yield protein deacetylation versus ADP-ribosylation will be elucidated. Aim 2 focuses on understanding how sirtuins recognize specific protein targets. To explore the biological functions of OAADPr, Aim 3 will involve the synthesis of nonhydrolyzable OAADPr analogs, followed by an examination of their ability to act as ligands, substrates and inhibitors against reported protein targets. The experiments in Aim 4 directly probe the cellular function of OAADPr. An assay for the quantitation of cellular OAADPr levels will be coupled with cell- based assays of genetically modified cells. The ability of OAADPr to promote gene silencing in yeast will be assessed. In mammalian cells, the ability of sirtuins and OAADPr to modulate the gating of the TRPM2 ion channel will be determined. Resveratrol, plant polyphenol known to harbor a variety of cardiovascular and neurological health benefits, was reported to be an activator of Sir2 enzymes. Here several hypotheses for the ability of resveratrol to "activate" SIRT1 will be examined in vivo and in vitro (Aim 2). Given the implications for cancer, aging and neurodegeneration, sirtuins have emerged as viable drug targets to treat these diseases. The knowledge gained from this study will provide the major steps towards the design and development of such small molecules.